The biological activity of conjugated linoleic acids (hereinafter CLA) has been well documented in a number of indications. Its effect as an anticarcinogenic agent was demonstrated in a rat mammary tumor model by Ha, et al., Cancer Res., 52: 2035s (1992), and in a mouse forestomach neoplasia model (Ha, et al., Cancer Res., 50: 1097 (1990). CLA has been found effective in attenuating allergic reactions mediated by type I or TgE hypersensitivity. As a nutritive supplement, CLA administration results in selective reduction in body fat, as disclosed in U.S. Pat. No. 5,554,646, and has a significant positive effect on feed conversion efficient as shown in U.S. Pat. No. 5,428,072.
Linoleic acid is an important component of biolipids, and comprises a significant proportion of triglycerides and phospholipids. It is an essential fatty acid, in that it is required in the diet for maintenance of healthy cells, but the body does not possess the enzymatic machinery to synthesis the fatty acid itself. Linoleic acid has 18 carbon atoms with double-bonds at positions 9 and 12. The conjugated forms of linoleic acid have the double bond positions shifted so that the double bond pairs are separated by a single methylene group. The rearrangement of the double bonds of linoleic acid to conjugated positions results in eight possible geometric isomers of 9,11 and 10,12 octadecanoic acid (c9,c11; c9,t11; t9,c11; t9,t11; c10,c12; c10,t12; t10,c12; and t10,t12. Other minor conjugated forms appear in nature and result from synthetic processes, namely, ct8,ct10 and ct11,ctl3 being the most prevalent.
A general mechanism for the isomerization of linoleic acid was described by J. C. Cowan in JAOCS, 72: 492 (1950). It is believed that the double bond is polarized by the result of a collision with an activating catalyst. The polarized carbon atom and its adjoining carbon are then free to rotate and the forces are such to make the deficient carbon atom essentially planar. When the system relieves forces set up as a result of the molecular collision, both cis and trans isomers are formed. More of the 10,12 and 9,11 isomers are formed than other species because of the thermodynamic stability of these forms. More severe conditions of heat, pressure, and polarity tend to drive isomerization further to the more stable trans,trans isomers, and cause redistribution of the double bonds with the appearance of significant quantities of the 8,10 and 11,13 forms.
One problem with aqueous alkali isomerization, which is the principal industrial process for producing CLA, is the formation of these multiple species. The reaction becomes uncontrolled and a significant proportion of the linoleic acid substrate is sacrificed to undesirable trans, trans isomers. For industrial use in drying oils where generalized polymerization between fatty acid strands is sought, it makes little difference which species of conjugated isomer predominate. However, in therapeutic or nutritional applications, the t10,c12 and c9,t11 isomers are believed to contain most, if not all, of the biological activity.
Other methods have been described utilizing metal catalysts, which are not highly efficient. Isomerization in these systems could be achieved more rapidly in the presence of higher molecular weight solvents. Kass, et al., J. Am. Chem. Soc., 61:4829 (1939) showed that replacement of ethanol with ethylene glycol resulted in both an increase in conjugation in less time. U.S. Pat. No. 2,350,583 and British Patent No. 558,881 (1944) achieved conjugation by reacting fatty acid soaps of an oil with an excess of aqueous alkali at 200–230 degrees C. and increased pressure. Among the processes known to cause isomerization in the absence of aqueous alkali, is a nickel-carbon catalytic method, as described in Radlove, et al., Ind. Eng. Chem., 38:997 (1946).
Processes have also been described for isomerization of polyethanoid fatty acids in their ester forms. U.S. Pat. Nos. 2,242,230 and 3,162,658 disclose methods in which the lower alkyl esters of linoleic acid are isomerized by catalysis with basic alcoholates, preferably sodium or potassium at moderate temperatures in the range of 100–140 degrees C. Typically these processes are used to generate industrial drying oils, and hence predominately utilize soy and corn oil is the starting material, in order to enhance polymerization when coated onto surfaces. These fatty acid ester compositions are not suitable for human or animal consumption because of high phosphidyl and other residue content. Purification by distillation, differential extraction, and the like removes the residues, but also causes further double bond rearrangements giving an unacceptable level of trans,trans CLA isomers, and intermolecular polymers.
The purified CLA utilized in prior feeding studies was obtained by small scale laboratory procedures involving production of CLA from highly purified linoleic acid. For example, Sullivan, J. Am. Oil Chemists' Soc., 53:359 (1976) describes a laboratory semi-pilot steam refining system made entirely of glass. While such systems are adequate for producing quantities of CLA for laboratory studies, or even clinical trials, they are not suitable for commercial scale bulk production. On the other hand, the large scale systems available to produce industrial quantities of CLA cannot be run inexpensively enough to produce material for bulk animal feeds. The degumming, refining, and dehydration steps necessary to obtain nutritionally safe edible CLA for livestock feeding are prohibitively complex and expensive.
Economical CLA-ester production in commercial quantities is a desirable objective in light of nutritional benefits observed on a laboratory scale. The advantages of an ester derivative rather than the free CLA fatty acids include resistance to oxidation, ease of manufacture according to the process of the present invention, palatability, and compatibility with lipid feed components.